Structural Plasticity and Functional Interactions of the Signaling Adapter NHERF

项目总结 许多参与细胞信号传递的蛋白质包含蛋白质-蛋白质相互作用模块的串联重复 属于PDZ(PSD95-Dlg1-Zo1)结构域家族，它识别以下短氨基酸基序 通常，但不是唯一的，位于蛋白质的C-末端。PDZ结构域的亲和力和特异性 对于正则(C-末端)的多肽配体已有广泛的研究。虽然有证据表明国际- 分子间的相互作用可以通过竞争的分子内相互作用来调节，结构 这种自身抑制效应的基础和激活机制还不是很清楚。要解决这个问题 问题，我们关注的是Na+/H+交换调节因子1(NHERF；也称为NHERF1或EBP50)，以及 参与调节细胞膜-细胞骨架界面重要信号事件的适配蛋白 上皮细胞。NHERF包含两个PDZ结构域和一个C末端Ezrin结合基序(EBM)，以及Long 无序的区域。这个项目的第一个目标是在结构/动力之间建立联系 NHERF的性质及其对生理相互作用伙伴的亲和力。基于荧光的结合分析， 快速混合和各种核磁共振技术，包括化学位移分析、顺磁松弛研究 和H-D交换，将提供对(I)NHERF识别多肽的机制的详细见解 生物相互作用伙伴的C末端衍生的配体，(Ii)结构和热力学基础 调节PDZ结构域的结合亲和力和特异性，以及(Iii)激活机制 由细胞骨架相互作用伙伴触发。第二个目的是阐明NHERF在调节中的作用 表皮生长因子受体(EGFR)的信号、内吞和降解是一种重要的 许多癌症的致癌驱动因素。细胞生物学研究确定了C-末端的潜在相互作用部位 NHERF的第一个PDZ结构域的EGFR的调节区。然而，还没有生物物理的 体外研究，以及这些相互作用的结构细节和生物学意义很差 明白了。我们试图通过使用核磁共振和荧光技术来填补这些知识空白(I) 阐明NHERF与细胞质调节相互作用的结构和能量基础 区域EGFR相互作用，(Ii)膜环境和效应器影响的生物物理研究 蛋白质对NHERF-EGFR相互作用的影响，以及(Iii)通过使用流式细胞术和细胞成像方法 探讨与NHERF的相互作用对EGFR内化、循环和/或降解的影响 人类癌细胞株。这些细胞生物学方法，与结构和机制相结合 从拟议的生物物理研究中获得对NHERF的激活及其与EGFR相互作用的见解， 将为NHERF在调节细胞稳定性、细胞定位和细胞中的作用提供详细的新见解 EGFR的促生长活性。

PROJECT SUMMARY Numerous proteins involved in cell signaling contain tandem repeats of protein-protein interaction modules belonging to the family of PDZ (PSD95-Dlg1-Zo1) domains, which recognize short amino acid motifs that are typically, but not exclusively, located at the C-terminus of a protein. The affinity and specificity of PDZ domains for canonical (C-terminal) peptide ligands has been studied extensively. While there is evidence that inter- molecular PDZ-peptide interactions can be modulated by competing intramolecular interactions, the structural basis of such autoinhibitory effects and the mechanism of activation are not well understood. To address this question, we focus on Na+/H+ exchanger regulatory factor 1 (NHERF; also known as NHERF1 or EBP50), an adapter protein involved in regulating important cell signaling events at the membrane-cytoskeleton interface of epithelial cells. NHERF contains two PDZ domains and a C-terminal ezrin-binding motif (EBM), as well as long disordered regions. The first aim of this project is to establish a linkage between the structural/dynamic properties of NHERF and its affinity for physiological interaction partners. Fluorescence-based binding assays, rapid mixing and various NMR techniques, including chemical shift analysis, paramagnetic relaxation studies and H-D exchange, will provide detailed insight into (i) the mechanism by which NHERF recognizes peptide ligands derived from the C-termini of biological interaction partners, (ii) the structural and thermodynamic basis of regulation of the binding affinity and specificity of the PDZ domains, and (iii) mechanisms of activation triggered by cytoskeletal interaction partners. The second aim is to elucidate the roles of NHERF in regulating signaling, endocytosis and degradation of the epidermal growth factor receptor (EGFR), an important oncogenic driver of many cancers. Cell-biological studies identified potential interaction sites in the C-terminal regulatory region of EGFR for the first PDZ domain of NHERF. However, there have been no biophysical studies in vitro, and the structural details and biological implications of these interactions are poorly understood. We seek to fill these gaps in knowledge (i) by using NMR and fluorescence techniques to elucidate the structural and energetic basis of interactions between NHERF and the cytoplasmic regulatory region EGFR interactions, (ii) biophysical studies of the influence of the membrane environment and effector proteins on NHERF-EGFR interactions, and (iii) by using flow cytometry and cellular imaging approaches to explore the impact of interactions with NHERF on internalization, recycling and/or degradation of EGFR in human cancer cell lines. These cell-biological approaches, combined with the structural and mechanistic insight into activation of NHERF and its interactions with EGFR gained from the proposed biophysical studies, will yield detailed new insight into the role NHERF in regulating the stability, cellular localization and cell growth-stimulating activities of EGFR.